| Bacillus anthracis is a spore-forming, rod-shaped, gram-positive bacterium. It is the causative agent of the disease anthrax of human and herbivores, including cutaneous, gastrointestinal and inhalational anthrax. Anthrax is one of the five supremacies zoonosis and is a great harm to stock farming and human's health. Besides, B.anthracis is used as a biological warfare agent and a terrorist weapon causing a serious threat. Because of anthrax's large influence of society, the relative research is always the hot point of life science.Now research on B.anthracis indicates that there are considerable genes contributed to virulence. These genes locate among plasmids and chromosome, and their products make up a sophisticated virulence network. The relative research hasn't been very clear now, so to identify additional virulent proteins will conduce to know more about B.anthracis and the pathogenesis of anthrax.Proteomics is not only a useful method to find out secrets behind vital phenomena systematically, but also an effective tool to screen something to study later. In this research, the data of proteomics was gathered and analyzed first, then a protein which might contribute to pathogenesis was selected and its functions were attempted to explore.If a pathogenic bacterium can infect host, it must be survival in the host and adapt to the change of environment, utilize energy effectively, and resist lots of antibiotic substances. Therefore proteins up-regulated in vivo were maybe associated with virulence. And the candidate proteins were screened by the strategy of comparative proteomics. Due to the difficulty in sample preparation, the proteomic data about B.anthracis in vivo were obtained by simulating in vitro. Such simulation was lack of the participation of host, and had a certain difference to real infection. Thus, it was tried to culture B.anthracis in the rabbit's intestinal tract. Rabbits are sensitive to B.anthracis, and gastrointestinal anthrax always be found at the ileocecal junction. Thus it was attempted to compare the proteins profiles of in vivo and vitro, and proteins playing a role in the adaptation of B.anthracis were screened. The vivo and vitro samples of supernatant proteins, cell wall proteins and whole cell proteins were extracted by different methods. Then these samples were thoroughly analyzed by means of two-dimensional gel electrophoresis combined with MALDI-TOF MS. About 144 differential expression protein spots had distinguished between in vivo and in vitro samples, and 125 spots were identified. To analyze the data, some features about the change of protein expression profiles in vivo were summarized. B.anthracis decreased general biosynthesis temporarily after entering the host, and so we could see many enzymes involved in synthesis down-regulating in vivo, such as threonyl-tRNA synthetase, prolyl-tRNA synthetase, long-chain-fatty-acid-CoA ligase. The up-regulated proteins have various functions. For example, DnaK and Sod-A are up-regulated in stress, and they showed the same trends in our results. The S-layer proteins are associated with surface structure and also expressed more in vivo. The change of these proteins indicated that after B.anthracis entering host, it decreased general biosynthesis and focused on the synthesis of certain protein which contributed to survive, implemente the balance of energy and the stabilization of its internal environment, then existed and developed in host.There was a putative S-layer protein which was an up-regulated protein in the supernatant sample and identified by MALDI-TOF MS. Its locus tag is BA3338, and its function is still unknown. Relative research shows that S-layer can protect cells, adhere to other cells, work as a molecular sieve, and provide the adhesion sides for the cell-associated exoenzymes. In pathogenic bacteria, the S-layer can even resist the aggression of host's immune system, and is considered to potential virulence factor. The studies on the S-layer proteins of B.anthracis indicate that they might be important targets for vaccines and drugs. And the research on the main S-layer protein of B.anthracis Sap and EA1 points out that they have distinct expression style and are regulated by network of virulence. Because of the importance of S-layer protein, the functions of BA3338 were tried to explore in this study.The proteins which might interact with BA3338 were first found by GST pull-down. BA3338 locates on the cell wall. It is supposed that it can interact with proteins of B.anthracis and proteins of host as well, playing different roles. So GST pull-down tests about BA3338 interacting with B.anthracis'whole cell proteins and BA3338 interacting with proteins of human blood serum and macrophage were carried out. To separate the suspicious specific adhesive protein, the beads were eluted and then the proteins of the elutriant after desalination were separated by 2D electrophoresis. This way could obtain more information and be easy to find out the disparate points, also easy to carry out by MS and avoid the interference of abundant proteins. Some suspicious specific adhesive proteins were found in the sample of BA3338 interacting with human serum protein. 11 samples were undergone and 5 of them were identified by MS, which was a higher ratio in such essays. It indicated that BA3338 might interact with Sap and EA1 of B.anthracis, as well as fibronectinâ… , IHRP and SP40/40 of host. The similar work about BA3338 and the proteins of macrophage had been finished, but none were found because the total quantity of extracted macrophage's protein samples was not sufficient.Since the results of GST pull-down just indicated possibilities, whether these interactions really exist needed checking. The interaction between BA3338 and Sap, EA1 and fibronectinâ… were tried to detect. The Sap and BA3338 were expressed with His tag, and the interaction between Sap and BA3338 was directly testified. EA1 hasn't been expressed and need continuing to overcome some difficulties. The interaction between BA3338 and fibronectinâ… was attempted to detect by Far Western. However, because of the interference of the product of vacant vector, it needs checking further after removing such interference.The result of GST pull-down screen indicated that BA3338 might be concerned with the infection of B. anthracis, so the indirect bactericidal tests of anti-BA3338 antibody were carried out. The antibodies were obtained by the immunization of rat. However, rats are insensitivity to B. anthracis and it was difficult to identify the effect of anti-BA3338 antibody because of the interference of rat's nonimmune serum. But the conspicuous inhibition of rat's serum to vegetative and spore form might be worthy to study further.
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